Unfortunately, it has increasingly been seen over the past several decades that employment of conventional chemical insecticides often leads to undesirable environmental consequences. Such consequences include toxicity to non-target organisms such as birds and fish, and human health hazards. Furthermore, pesticide management in the United States and elsewhere in the world is becoming increasingly complicated due to the evolution of insect resistance to classical chemical pesticides. Despite over 10 billion dollars being spent each year to control phytophagus insects, global losses in the food supply due to insects is still estimated to be about 20 to 30 percent (See, Oerke, Estimated crop losses due to pathogens, animal pests and weeds, 72-78 in Crop production and crop protection: Estimated losses in major food and cash crops (Elsevier, Amsterdam 1994)). There remains, therefore, an urgent need to develop or obtain substances that can be used safely in the fight against insect pests.
Over the past several years, there have been proposed a number of “environmentally friendly” strategies to combat highly resistant insect pests such as certain species of cotton bollworm (e.g., Helicoverpa zea).
One recently introduced approach to insect management is the production of transgenic crops that express insecticidal toxins, such as engineered potato and cotton crops that express protein toxins from the soil bacterium Bacillus thuringiensis (Estruch, J. J. et al., Transgenic plants: An emerging approach to pest control, Nature Biotechnology 15, 137-141, 1997).
A variation of this strategy is the release of insect-specific viruses that have been genetically engineered to express insecticidal neurotoxins (Cory, J. S. et al., Field trial of a genetically improved baculovirus insecticide, Nature 370, 138-140, 1994). Baculoviruses, for example, are arthropod-specific viruses with no member of the baculovirus family known to infect either vertebrates or plants. The infectivity of some baculoviruses is restricted to a few closely related species within a single family of lepidopterous insects (moths and butterflies) (See, e.g., U.S. Pat. No. 5,639,454). Some baculoviruses, such as the beet armyworm nuclear polyhedrosis virus, target only a single species. As a result of their high degree of specificity, baculoviruses have long been envisaged as potential pest control agents and were first used as such in the 1970s. Their specificity means that baculoviral insecticides complement natural predators, rather than replacing them, as is the case with many chemical insecticides. However, to date, baculoviruses have met with only limited commercial success. Most naturally occurring baculoviruses take 4-7 days to kill their hosts, with some species taking considerably longer. During this time the insect continues to feed and cause crop damage, thus limiting the ability of baculoviral insecticides to compete with chemical agents.
This shortcoming has been addressed by engineering recombinant baculoviruses that express insect-specific neurotoxins. Expression of heterologous insect toxins not only reduces the time interval between virus application and insect death, but also reduces the mean feeding time (Prikhod'ko et al., Effects of simultaneous expression of two sodium channel toxin genes on the properties of baculoviruses as biopesticides, Biological Control 12, 66-78, 1998). Importantly, introduction of genes for insect-selective toxins does not alter the intrinsic infectivity of the baculovirus or its natural host range (Black et al., Commercialization of baculoviral insecticides, in The Baculoviruses (ed. Miller, L. K.) 341-387 (Plenum Press, New York, USA, 1997)).
New approaches to insect-pest management have stimulated interest in peptide toxins from the venoms of animals, particularly spiders and scorpions, that prey on insect species.
Zlotkin et al., An Excitatory and a Depressant Insect Toxin from Scorpion Venom both Affect Sodium Conductance and Possess a Common Binding Site, Arch. Biochem. and Biophysics 240, 877-887, 1985), disclose two insect selective toxins from the venom of the scorpion Leiurus quinqestriatus, one of which induced fast excitatory contractive paralysis of fly larvae while the other induced slow depressant flaccid paralysis, with both affecting sodium conductance in the neurons. Likewise, Canadian patent 2,005,658 (issued: Jun. 19, 1990 to Zlotkin et al.) discloses an insecticidally effective protein referred to as “LqhP35” derived from the scorpion Leiurus quinquestriatus hebraeus.
A number of investigators have also recognized spider venoms as a possible source of insect-specific toxins for agricultural applications (See, Jackson et al., Ann. Rev. Neurosci. 12, 405-414 (1989)). For example, U.S. Pat. Nos. 4,855,405 (issued: Aug. 8, 1989 to Yoshioka et al.) and 4,918,107 (issued: Apr. 17, 1990 to Nakajima et al.) both disclose glutamate-receptor inhibitors obtained from the venom of spiders as possible insecticidal agents. In U.S. Pat. Nos. 5,457,178 (issued: Oct. 10, 1995), 5,695,959 (issued: Dec. 9, 1997), and 5,756,459 (issued: May 26, 1998), Jackson et al. disclose a family of insecticidally effective proteins isolated from the venom of the spiders Filistata hibernalis (a common house spider) and Phidippus audax (a “jumping spider”).
A particular group of spiders which has generated considerable investigative interest are the funnel-web spiders. WO 89/07608 (published: Aug. 24, 1989, Cherksey et al.) discloses low molecular weight factors isolated from American funnel-web spider venoms which reversibly bind to calcium channels. Adams et al., Isolation and Biological Activity of Synaptic Toxins from the Venom of the Funnel Web Spider, Agelenopsis aperta, in Insect Neurochemistry and Neurophysiology, Borkovec and Gelman (eds.) (Humana Press, New Jersey, 1986) teaches that multiple peptide toxins which antagonize synaptic transmission in insects have been isolated from the spider Agelenopsis aperta. In WO 93/15108, a class of peptide toxins known as the ω-atracotoxins are disclosed as being isolated from the Australian funnel-web spiders (Araneae:Hexathelidae:Atracinae) by screening the venom for anti-Helicoverpa (“anti-cotton bollworm”) activity. Such toxins are disclosed to have a molecular weight of approximately 4000 amu, to be of 36-37 amino acids in length, and capable of forming three intrachain disulfide bridges. One of these compounds, designated ω-ACTX-Hv1 has been shown to selectively inhibit insect, as opposed to mammalian, voltage-gated calcium channel currents (Fletcher et al., The structure of a novel insecticidal neurotoxin, ω-atracotoxin-Hv1, from the venom of an Australian funnel web spider, Nature Struct. Biol. 4, 559-566 (1997)). Homologues of ω-ACTX-Hv1 have been isolated from the Blue Mountain funnel-web spider Hadronyche versuta (See, Wang et al., Structure-function of ω-atrocotoxin, a potent antagonist of insect voltage-gated calcium channels, Eur. J. Biochem. 264, 488-494 (1999)).
While some of the insecticidal peptide toxins isolated so far from scorpions and spiders offer promise, there still remains a significant need for compounds which display a wide differential in toxicity between insects and non-insects, and yet have significant insecticidal activity and a quick action.
The present inventors isolated, and structurally and functionally characterized, a novel insecticidal toxin, designated ω-atracotoxin-Hv2a, from the venom of the Australian funnel-web spider H. versuta. This ω-atracotoxin-Hv2a which is the subject of U.S. Pat. No. 6,583,264 B2 toxin is a highly potent and specific antagonist of insect calcium channels. The ω-atracotoxin-Hv2a toxin of the invention disclosed in U.S. Pat. No. 6,583,264 B2. shows no significant sequence similarity to any previously isolated insecticidal toxins, and no sequence or structural homology with the omega-atracotoxin -Hv1 family of insecticidal toxins previously isolated from H. versuta (See, Atkinson et al., Insecticidal toxins derived from funnel web spider (Atrax or Hadronyche) spiders, PCT/AU93/00039 (WO 93/15108) (1993); Fletcher et al., The structure of a novel insecticidal neurotoxin, ω-atracotoxin-HV1, from the venom of an Australian funnel web spider, Nature Struct. Biol. 4, 559-566 (1997); Wang et al., Structure-function studies of ω-atracotoxin, a potent antagonist of insect voltage-gated calcium channels, Eur. J. Biochem. 264, 488-494 (1999)).
The invention disclosed in U.S. Pat. No. 6,583,264 B2 is directed to the use of ω-atracotoxin-Hv2a, or the gene coding for the toxin, as a biopesticide, either alone or in combination with other insecticidal toxins or genes thereof. It further teaches the use of the toxin, or the gene coding for the toxin, as a screen for natural or non-natural compounds that specifically inhibit insect calcium channels. Furthermore, the patent's disclosure provides in the determination of the toxin's three-dimensional structure, a model for developing non-peptidic mimics of the toxin that could be used as foliar pesticide sprays.
As disclosed in the patent, there is provided a polypeptide toxin that is toxic to adult and/or larval insects having a molecular mass of approximately 4,478 Daltons and a length of 45 amino acid residues. The polypeptide is capable of forming three intrachain disulfide bonds. Activity of polypeptide as a toxin has been demonstrated by rapid paralysis of insects and/or potent inhibition of whole-cell calcium currents in isolated insect neurons. Phylogenetic specificity of the polypeptide toxin was demonstrated by minimal activity in rat or chicken nerve-muscle preparations and/or minimal antagonism of calcium channel currents in isolated rat neurons.
The preferred toxin as disclosed in U.S. Pat. No. 6,583,264 B2 is omega -atracotoxin-Hv2a (SEQ ID NO: 1), abbreviated as omega-ACTX-Hv2a or ω-ACTX-Hv2a, as defined below:
SEQ ID NO:1:Leu-Leu-Ala-Cys-Leu-Phe-Gly-Asn-Gly-Arg-Cys-Ser- Ser-Asn-Arg-Asp-Cys-Cys-Glu-Leu-Thr-Pro-Val-Cys- Lys-Arg-Gly-Ser-Cys-Val-Ser-Ser-Gly-Pro-Gly-Leu- Val-Gly-Gly-Ile-Leu-Gly-Gly-Ile-Leu (LLACLFGNGR CSSNRDCCEL TPVCKRGSCV SSGPGLVGGI LGGIL)